Chicken anaemia virus (CAV) is a member of the Circoviridae family. The Circoviridae include a number of plant and animal viruses that are characterised by the possession of a single stranded, negative-sense, circular DNA genome. There is minimal similarity in the genomic sequence and organisation between CAV and the other characterised animal circoviruses: Psittacine Beak and Feather Disease Virus (PBFDV), Pigeon Circovirus and Porcine Circoviruses (PCV) 1 and 2. TT viruses (TTV) have recently been identified in human hosts and other species as a heterogeneous cluster of single stranded, negative-sense, circular DNA viruses. Sequence analysis of this group of viruses has demonstrated greatest overall homology to CAV and others have recently proposed the classification of the TTV, SANBAN, YONBAN, TLMV (TTV Like Mini Viruses) and CAV viruses as the Paracircoviridae, however, the phylogeny remains an area of active revision. The highest sequence homology to CAV is seen in the non-coding region and between open reading frame (ORF2) of TTV and VP2 of CAV. The high level of sequence conservation between CAV and TTV suggests VP2 may play a critical role in viral infection and pathogenesis.
CAV encodes only three proteins, with overlapping ORFs in three frames. ORF3 encodes the major 45-52 kDa capsid protein VP1, ORF2 encodes the 11-13 kDa VP3 that has demonstrated apoptotic activity in transformed cell lines, and ORF1 encodes a 28 kDa non-structural protein VP2 with unknown function. VP2 is expressed at barely detectable levels during infection, but has been shown to be essential for viral infection and replication in cells. The low level of expression of VP2 is consistent with a non-structural, regulatory protein involved in viral replication and infection.
CAV pathogenesis is characterised by immunosuppression and pancytopaenia arising from panmyelophthisis and thymocyte depletion. Immunosuppression results in increased rates of morbidity and mortality associated with coinfections and vaccination failure in CAV infected chicks. CAV infection is directly cytotoxic to two distinct T-cell populations of the thymus and spleen. Thymic infection involves immature lymphoblastic precursors, whereas splenic infection is of mature T-lymphocytes that are highly activated. There is a second indirect-component of immunosuppression found in uninfected immune effector cells. Reductions in macrophage and APC effector functions and B cell antigenic responses have been documented. Limited cytokine profiles from infected cells are suggestive of a basis for generalised indirect immunosuppression. There is a reduction in interleukin 2 (IL-2), interferon gamma (IFNγ), lymphocyte stimulation index, IL-1, T-cell growth factor activity and Fc receptor levels in lymphocytes of infected birds. The molecular basis for viral modulation of cytokine profiles and indirect immunosuppression is unknown.
Preliminary comparisons of the CAV VP2 sequence to sequences available in the Genbank database suggested similarity to a number of eukaryotic receptor PTPases (R-PTPase alpha). Database searches identified the human placental, rat, mouse and chicken R-PTPase alpha precursors as homologous to the CAV VP2 sequence. Reversible protein phosphorylation is universal in the regulation of cellular processes, including metabolism, gene regulation, cell cycle control, cytoskeletal organisation and cell adhesion. The PTPase family is highly diverse and includes the eukaryotic receptor-like transmembrane proteins and soluble cytosolic proteins, as well as bacterial PTPases, such as the YopH PTPase from pathogenic Yersinia, and a viral PTPase VH1 found in Vaccinia virus, a member of the Poxyiridae. During Vaccinia virus infection the VH1 protein blocks interferon γ signalling thereby evading the immune response to virus infection. The role of the VH1 PTPase in infection, although currently the only viral PTPase with a characterised in vivo function, does highlight the potential for virus encoded PTPases to be involved in mechanisms of immune evasion and virus persistence.
Commercial poultry producers require a chicken anaemia virus (CAV) vaccine that will reduce the economic losses incurred through both clinical and subclinical infections. The elimination of subclinical disease in adult birds associated with CAV infection requires overcoming immunosuppression due to infection. CAV infection is of greatest economic significance in broiler flocks. Both clinical and subclinical infections impact on commercial broiler performance and profitability. Whilst clinical infection produces a more marked reduction in performance parameters, subclinical infection is responsible for a greater degree of financial loss as it is of higher incidence. There is a strong need for a vaccine suitable for pullets, broilers and breeders. Such a vaccine may be administered to birds at the point of lay and therefore must be safe in the event of vertical transmission to embryos.
The development of a CAV vaccine has international applications. Chicken anaemia virus (CAV) has a worldwide distribution based on serological surveillance, and is endemic in both SPF and commercial chicken flocks, with the exception of Australian SPF flocks. Countries from which CAV isolates have been characterised and their complete genome sequences published include Germany, UK, USA, Japan, Australia, and the Netherlands. All isolates are classified within a single serotype based on cross reactivity in immunofluorescence and neutralisation tests utilising polyclonal antiserum. Genome sequence conservation is a key feature of all CAV isolates. All field isolates demonstrate equivalent pathogenicity in experimental infection and any variation in the morbidity and severity of disease with CAV exposure is attributed to a range of interacting, epidemiological factors. Viral dose is the key determinant of the severity of CAV induced disease in the field. It is expected that live attenuated vaccines developed from any one isolate will be protective in poultry flocks internationally.
An attenuated CAV strain should be infectious whilst having reduced pathogenicity. Clinical disease is best characterised in the literature in birds infected at 1 day old. Clinical disease in chicks infected at 1 day of age is characterised by weakness, depression, stunting and anaemia. By 7 days post infection, there is a transient but severe, peracute anaemia due to destruction of erythroblastoid cells and immunodeficiency due to depletion of cortical lymphocytes. Severe bone marrow hypoplasia, thymic and lymphoid atrophy and thrombocytopaenia are apparent at 14-21 days post infection. Petecchial and ecchymotic haemorrhages develop due to a primary coagulopathy. Immunosuppression is a significant feature of CAV induced disease and secondary infections are common. CAV affected birds have an increased incidence of malignant oedema, gangrenous dermatitis, colibacillosis and pulmonary aspergillosis. The recovery phase extends from 14-35 days post infection. Erythrocytopoiesis precedes granulocytopoiesis during recovery. At 16 days post infection there are a high proportion of circulating immature erythrocytes, thrombocytes and granulocytes, and the haematocrit is completely restored by 28 days post infection. The thymus is repopulated by the third wave of migrating lymphocytes at 21 days.
CAV affected birds develop a severe anaemia of myelophthisis. The haematocrit is less than 27%, and typically between 9-23% (normally in chicks 7-14 days of age it is 32-37.5%). Cyanosis is evident in the non-feathered integument and on mucosal membranes. There is a leukopaenia attributable to a heterocytopaenia and lymphopaenia. Prolonged clotting times are associated with petecchial and ecchymotic haemorrhages observed over the integument, skeletal muscle, mucosa of the proventriculus and rarely the pericardium.
The bone marrow appears yellow to white and watery in texture due to panmyelophthisis and compensatory adipocyte hyperplasia. This is most obvious in the proximal femoral medullary cavity.
The thymus undergoes severe atrophy. Affected thymuses have a quantifiable reduction in weight and a diameter of 2-4 mm. They appear red-brown instead of grey due to a reduction in parenchymal lymphocyte populations, hyperplasia of reticular cells and hyperaemia of the tissue.
There is a generalised depletion of lymphoid follicular components of all tissues. The bursa of Fabricius undergoes transient, moderate atrophy but is not swollen or oedematous. Bursal atrophy can be mild to unapparent in clinically affected chicks.
The liver, kidneys and spleen are diffusely discoloured and swollen at 14 days post infection.
Focal, dermal haemorrhagic lesions are most prominent on the wings, but also involve the head, rump, sides of thorax and abdomen, thighs, legs and feet. The lesions progress to large ulcers with a serosanguinous extravasation due to ischaemic necrosis of the overlying dermis. A purulent exudate develops in association with secondary infections. The lesions are prone to complicating abrasive and mutilation injury in the environment of the commercial broiler rearing unit.
An experimental model for CAV pathogenesis is required for the assessment of attenuation. Such a model does not need to represent the full spectrum of pathology observed in field infection but must demonstrate attenuation under conditions that produce most severe pathology. Yolk sac inoculation of 7 day embryos with high doses of virus is the most stringent model available. This model best approximates the field situation in which naïve breeder birds at the point of lay are exposed to CAV and transmit virus transovarially. Chicks infected by vertical transmission have the highest rates of morbidity (100%) and mortality (10-70%) and the pathology is of greatest severity. Extensive studies of the pathology of embryos experimentally infected at 7 days by yolk sac inoculation have not been reported in the literature.
Chickens of all ages are susceptible to CAV infection, however there is an age-specific resistance to the development of disease in chickens older than 14 days. Embryos and 1 day old chicks have the highest disease susceptibility. Age resistance may relate to the developing capacity of the bird to produce a serum neutralising humoral response. Co-infection with synergistic avian pathogens such as IBDV will eliminate age-related resistance and will result in outbreaks of acute severe disease in older birds.
The majority of commercial breeder flocks have been exposed to CAV and have long lasting neutralising humoral immunity. Antibody persists for at least 20 weeks after seroconversion. Serological surveys of breeder flocks typically demonstrate 97.5-100% of birds remain seropositive over an extended period post infection. Maternal antibody is important in protection against clinical disease in chickens up to 2 weeks of age, and persists until 3 weeks of age. The decay of maternal antibody follows a linear relationship and has a half life of approximately 1 week. Low levels of maternal antibody are effective in preventing clinical disease with infection. The majority of hatchlings derived from immune breeder flocks are infected horizontally following the waning of maternal antibody, develop subclinical disease and seroconvert between 8-12 weeks post infection. In an exposed flock, approximately 10% of breeders will be seronegative at any point post exposure. A minor proportion of chickens are infected vertically and excrete high titres of virus acting as the source of horizontal infection for other hatchlings. There may be between 16 and 25% birds sub-clinically affected in the progeny of immune breeder flocks. Vaccination will therefore improve performance even in flocks with endemic CAV and persistent neutralising humoral immunity.
The present inventors have developed live attenuated CAV and CAV DNA capable for use in vaccines suitable for the inoculation of pullets, broiler and breeder flocks, based on the identification of the function of the VP2 as a novel protein tyrosine phosphatase and the identification of regions of its sequence required for full function.